Rad51 protein controls Rad52-mediated DNA annealing - PubMed (original) (raw)

Rad51 protein controls Rad52-mediated DNA annealing

Yun Wu et al. J Biol Chem. 2008.

Abstract

In Saccharomyces cerevisiae, Rad52 protein plays an essential role in the repair of DNA double-stranded breaks (DSBs). Rad52 and its orthologs possess the unique capacity to anneal single-stranded DNA (ssDNA) complexed with its cognate ssDNA-binding protein, RPA. This annealing activity is used in multiple mechanisms of DSB repair: single-stranded annealing, synthesis-dependent strand annealing, and cross-over formation. Here we report that the S. cerevisiae DNA strand exchange protein, Rad51, prevents Rad52-mediated annealing of complementary ssDNA. Efficient inhibition is ATP-dependent and involves a specific interaction between Rad51 and Rad52. Free Rad51 can limit DNA annealing by Rad52, but the Rad51 nucleoprotein filament is even more effective. We also discovered that the budding yeast Rad52 paralog, Rad59 protein, partially restores Rad52-dependent DNA annealing in the presence of Rad51, suggesting that Rad52 and Rad59 function coordinately to enhance recombinational DNA repair either by directing the processed DSBs to repair by DNA strand annealing or by promoting second end capture to form a double Holliday junction. This regulation of Rad52-mediated annealing suggests a control function for Rad51 in deciding the recombination path taken for a processed DNA break; the ssDNA can be directed to either Rad51-mediated DNA strand invasion or to Rad52-mediated DNA annealing. This channeling determines the nature of the subsequent repair process and is consistent with the observed competition between these pathways in vivo.

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Figures

FIGURE 1.

FIGURE 1.

Rad51 inhibits Rad52-mediated DNA annealing. The reactions were conducted as described under “Experimental Procedures” and are illustrated schematically at the top of the figure. Rad51 inhibits Rad52-mediated annealing of complementary ssDNA (A) and RPA-ssDNA complexes (B). A representative gel of Rad52-promoted DNA annealing is shown in the left panel, and the quantification is shown in the right panel. The reactions contained Rad52 alone (-51, +52; black squares), both Rad51 and Rad52 (+51, +52; blue triangles), neither Rad51 nor Rad52 (-51, -52; gray squares), and Rad51 alone (+51, -52; green triangles). ATP was omitted from the experiment represented by red circles. The results are the averages obtained from at least three independent experiments, and the error bars represent one standard deviation (where absent, the error bars are smaller than the symbol). C, time course of Rad52-mediated DNA annealing as a function of Rad51 concentration (gels not shown). The extent of DNA annealing at 4 min is plotted versus the Rad51 concentration and is shown in D as blue squares; the red triangles are from experiments where Rad51 and Rad52 were incubated together and added simultaneously. The results are the averages obtained from at least two independent experiments, and the error bars represent the variation.

FIGURE 2.

FIGURE 2.

Inhibition of Rad52-dependent DNA annealing by Rad51 is species-specific. The reactions were carried out as described under “Experimental Procedures” except RecA (134 n

m

, blue lines) or SSB (50 n

m

, red lines) was substituted for Rad51 or RPA, respectively. Control reactions containing RPA only or RPA and RecA are shown as gray squares and green triangles, respectively. The results are the averages obtained from two independent experiments, and the error bars represent the variation.

FIGURE 3.

FIGURE 3.

Rad52-promoted DNA annealing between RPA-ssDNA complexes and Rad51-ssDNA filament is reduced. Protein-ssDNA complexes were formed by separately incubating RPA (30 n

m

) with 5′-32P-ssDNA (W) (400 n

m

), and the protein indicated with ssDNA (C) (400 n

m

) in DNA annealing buffer with 1 m

m

ATP for 5 min. Annealing was initiated by mixing equal volumes of the two complexes, and then Rad52 was added at 1 min (triangles). The reactions lacking Rad52 are shown as squares. The reactions containing either E. coli RecA (134 n

m

), RPA (30 n

m

), human Rad51 (134 n

m

), and yeast Rad51 (134 n

m

) are represented by the red, black, green, and blue symbols, respectively. The results are the averages obtained from at least two independent experiments, and the error bars represent reaction variation.

FIGURE 4.

FIGURE 4.

The Rad51 nucleoprotein complex is the most effective inhibitor of Rad52-mediated DNA annealing. The experiments were carried out as described under “Experimental Procedures” except that in A Rad51 was preassembled on heterologous dsDNA (400 n

m

) at 37 °C for 5 min before addition to the RPA-ssDNA complexes. DNA was 100 bp in length. B, experiments were carried out as in A except that Rad51 was preassembled on heterologous ssDNA (400 n

m

). DNA was 100-nucleotides in length. In both panels, Rad52 only (blue squares) and with Rad51 (blue triangles), heterologous DNA (green squares), and Rad51-DNA complex (green triangles) with ATP are shown. The experiments carried out in the presence of ATPγS in lieu of ATP are represented by the open symbols: +Rad51, orange triangles; +heterologous DNA, red squares; and +Rad51 and DNA (both), red triangles. The results are the averages obtained from at least two independent experiments, and the error bars represent the variation (where absent, the error bars are smaller than the symbol).

FIGURE 5.

FIGURE 5.

Rad59 alleviates the inhibitory effect of Rad51 in Rad52-mediated DNA annealing. Experiments were carried out as described under “Experimental Procedures.” RPA-complexed ssDNA was preincubated without or with Rad51 (134 n

m

), and the reactions were initiated by the addition of Rad52 (40 n

m

) and/or Rad59 (40 or 80 n

m

). The reactions either lacking or containing Rad51 are shown in black and blue, respectively. The reaction containing Rad52 alone (black open squares), Rad51 followed by Rad52 (blue open triangles), Rad51 followed by Rad52 and 40 n

m

Rad59 (blue solid triangles), and Rad51 followed by Rad52 and 80 n

m

Rad59 (blue diamonds) are shown. The results are the averages obtained from at least three independent experiments, and the error bars represent one standard deviation. Control reactions containing Rad59 only (80 n

m

; green solid circles), Rad51 and Rad59 (green open circles), and Rad52 and Rad59 (black solid squares) are shown. The results are the averages obtained from at least two independent experiments, and the error bars represent the variation (where absent, the error bars are smaller than the symbol).

FIGURE 6.

FIGURE 6.

Rad51 directly interacts with Rad59 protein in vitro. Pulldown experiments using magnetic Ni-NTA beads were carried out in the absence of DNA as described under “Experimental Procedures.” Proteins that remained free in the solution are shown in the left panel (unbound). Proteins retained on the beads were eluted by 300 m

m

imidazole and are shown in the right panel (bound). NaCl concentration in the initial binding reaction is indicated at the top.

FIGURE 7.

FIGURE 7.

A model for selection among the homologous recombination pathways of DSB repair. The _RAD51_-dependent and -independent recombination pathways are represented by two biochemical reactions: Rad51-mediated DNA strand exchange and Rad52-mediated DNA annealing. Both pathways share the common step of DSB resection and RPA binding to the 3′-ssDNA tails (step 1). The species-specific interaction between RPA and Rad52 protein recruits Rad52 and Rad59 to the RPA-ssDNA complex (step 2). In the _RAD51_-independent pathway (step 3, right arrow), Rad52 promotes annealing of RPA-ssDNA with a complementary sequence from the other end of the processed DSB. Rad59 plays an important role at this step by enhancing DNA annealing activity of Rad52 and counteracting the inhibitory effect of Rad51 protein. In the _RAD51_-dependent pathway (step 3, left arrow), with the help by Rad52 (and Rad55-Rad57), Rad51 displaces RPA and Rad52 from ssDNA to form the presynaptic complex; formation of the Rad51 nucleoprotein filament strongly inhibits Rad52-mediated DNA annealing. DNA strand invasion and exchange with homologous DNA duplex follow (step 4). Next, Rad51 protein is stripped off DNA by Rad54 protein, and DNA replication initiates from the invading strand (step 5). After the newly synthesized DNA is unwound from the displacement-loop (D-loop) intermediate (step 6, left), it anneals to the second processed end of the DSB in a Rad52-dependent manner, aided by Rad59 (step 7, left). Further DNA synthesis, branch migration, cleavage, and ligation complete repair of the DSB (step 8, left). Alternatively, the second end of the DSB can be directly annealed to the D-loop by Rad52-Rad59 proteins (step 6, right). After DNA replication, branch migration, and DNA ligation, the double Holliday junction (dHJ) structure is formed (step 7, right). Resolution of the double Holliday junction structure completes repair of the DSB (step 8, right).

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